ABSTRACT There are currently no reliable and objective urine-based, rapid-detection, point-of-care tests for mosquito-borne diseases. In this Phase 1 proposal we will develop and optimize the U-TEST, a test for urine samples that can distinguish between infections of multiple mosquito-borne viruses (MBVs) including Zika virus (ZIKV), Dengue virus (DENV), Chikungunya virus (CHIKV), and Yellow Fever virus (YFV). U- TEST will not require trained personnel, expensive equipment, or a centralized laboratory, and it is a rapid, low-cost option with high specificity that can be done in the field without electrical power and using room temperature urine without centrifugation. We have completed initial testing of samples from a patient cohort in a current mosquito-borne virus infection epicenter in partnership with Paramedics for Children International (PFCI) at a clinical facility in Copn Ruinas, Honduras. This clinic treats over 5,000 patients a year, and sees patients daily with mosqui- to-borne diseases. We have already received 93 urine and blood samples from PFCI that we will use to val- idate and optimize the U-TEST. We will run 186 U-TEST in the urine and blood samples and validate the positive and negative results with those results obtained with quantitative reverse transcription PCR (qRT- PCR) that has already been completed and discussed in the preliminary data section. Dr. Chancellor is an expert in designing urine-based biomarker assays and has an established track- record of translating and commercializing evidence-based laboratory inventions to the clinic. The intellectual property of the technology being developed in this SBIR was developed by two of the co-investigators, Drs. Michael Chancellor and Laura Lamb, and is exclusively owned by Lipella with global rights. This project is highly innovative and, to our knowledge, this is the first field based assay that can use simple voided urine that does not require centrifugation or refrigeration. We have the necessary knowledge and proficiency to be successful in the on-time completion of all the tasks of this grant. Upon successful completion of this Phase 1 project, we will have completed proof-of-concept assay kit that will be ready for field testing and regulatory validation. These results will lead to a well-designed Phase 2 proposal to assess the sensitivity, specificity, and predictive value of the diagnostic test in the real world clinical setting, and hopefully the method can be adopted in a larger US regulatory trial for commercialization of the proprietary U-TEST test.